Inflammatory disorders presently account for a significant percentage of debilitating diseases. Chronic conditions, such as rheumatoid arthritis, systemic lupus, psoriasis, and possibly atherosclerosis, stem from inflammatory reactions in the joints, skin and blood vessels. It is now apparent that a central role in the inflammatory reaction is the production of phospholipid metabolites called eicosanoids. It is generally accepted that in most tissues the synthesis of the eicosanoids is limited by the availability of arachidonic acid (AA) which is liberated from esterified stores in complex lipids. The liberation of AA is accomplished by the activity of phospholipases.
Phospholipase A.sub.2 (PLA2; EC3.1.1.4) catalyzes the release of fatty acids from the sn.sup.2 position of 1,2-diacyl-sn-glycero-3-phosphocholines. The best characterized varieties are the digestive enzymes secreted as zymogens in the pancreas of mammals. Amino acid sequences and cDNAs have been cloned for pancreatic PLA.sub.2 enzymes from a variety of mammals. See, e.g., O'Hara et al. (1976) J Biochem 99:733-739; Dufton et al. (1983) Eur J Biochem 137:537-544; Grataroli et al. (1982) Eur J Biochem 122:111-117. These mammalian PLA.sub.2 enzymes have a close homology to venom phospholipases of snakes and bees. Dufton et al., supra. In particular, the key active site residues and the alignment of cysteines appear to be highly conserved. X-ray crystallographic studies of bovine pancreatic PLA.sub.2, along with several venom enzymes, have led to the development of detailed models for PLA.sub.2 enzyme structure and mechanism of action. See, e.g., Renetseder et al. (1985) J Biol Chem 206:11627-11634. Both pancreatic and venom PLA.sub.2 have been shown to be proinflammatory. Pruzanski et al. (1986) J Invest Dermatol 86:380-383. An additional digestive PLA.sub.2 has been isolated from pig intestine and a partial amino acid sequence deduced. Verger et al. (1982) Biochemistry 21:6883-6889.
The structure of pancreatic PLA.sub.2 has been used as a model for designing novel PLA.sub.2 inhibitors. This approach, however, has not led to the design of a drug which has proved effective in inhibiting inflammation in vivo.
If PLA.sub.2 plays a central role in mammalian inflammatory disease, however, it probably is not through any of the digestive forms in most instances. Rather, analogous PLA.sub.2 enzymes, referred to as "cellular" PLA.sub.2 enzymes appear to be the likely regulator of AA release during the onset of inflammation. Unfortunately, these cellular PLA.sub.2 enzymes are not well understood. This is due to the fact that they are difficult to obtain in quantity and require more extensive purification than the digestive forms of PLA.sub.2.
Cellular forms of PLA.sub.2 have been isolated from a wide variety of mammalian tissues and cell types, including brain (Gray & Strickland, 1982, Can J Biochim 60:108-117), liver (DeWinter et al., 1982, Biochim Biophys Acta 712:332-341), lung (Franson et al., 1982, Lung160:275-284; Garcia et al., 1975, Biochim Biophys Res Comm 64:128-135; Sahu & Lynn, 1977, Biochim Biophys Acta 489:307-317), intestine (Verger et al., 1982, Biochemistry 21:6883-6889), spleen (Teramoto et al., 1983, J Biochim 93:1353-1360), macrophages (Trotter & Smith, 1986, Neurochem Res11:349-361; Lanni & Franson, 1981, Biochim Biophys Acta 658:54-63; Vadas & Hay, 1980, Life Sciences 26:1721-1729; Vadas et al., 1981, Nature 293:583; Wightman et al., 1981, Biochim J 200:441-444; Franson et al., 1973, Biochim Biophys Acta 296:365-373), leukocytes Traynor & Authi, 1981, Biochim Biophys Acta 665:571-577; Franson et al., 1977, Biochim J 167:839-841), erythrocytes (Kramer et al., 1978, Biochim Biophys Acta 507:381-394), ascitic fluid (Forst et al., 1986, Biochemistry 25:8381-8385), chondrocytes (Chang et al., 1986, J Immunol 136:1283-1287), and, platelets (Hayakawa et al., 1988 J Biochem 103:263-266; Hayakawa et al., 1987, J Biochim 101:1311-1314; Jesse & Franson, 1979, Biochim Biophys Acta 575:467-470; Apitz-Castro et al., 1979, Biochim Biophys Res Comm 91:1, 63-71). For a review, see Van Den Bosch (1980) Biochim Biophys Acta 604:191-246. See also, commonly owned U.S. patent application Ser. No. 946,557, filed Dec. 24, 1986.
Of particular interest is the isolation of a PLA.sub.2 from inflammatory exudates, such as the synovial fluid of rheumatoid arthritis patients. Stefanski et al., (1986) J Biochim 100:1297-1303; Vadas et al. (1985) Life Sciences 36:579-587; Vadas & Pruzanski (1984) Adv Inflammation Res 7:51-59; Vadas et al. (1981) Nature 293:583-585; Pruzanski et al. (1985) J Rheumatol 12:211-216; Silverman et al., American Rheumatism Ass'n: 51st Annual Scientific Meeting (Jun. 9-13, 1987, Washington, D.C.); Pruzanski et al., ibid.
Of these various cellular enzymes, the reports of their activity differ in size, pH optima, substrate specificity, Ca.sup.++ requirement, form (soluble vs. membrane-associated), and abundance. Since no complete protein sequences have been publicly reported for these isolates (partial sequences published by verger et al., 1982, supra; Forst et al., 1986, supra; Hayakawa et al., 1987, supra; and Hayakawa et al., 1988 supra), it is difficult to say which, if any, of these isolates represent the same enzymes. Moreover, it is difficult to completely discriminate between PLA.sub.1 and PLA.sub.2 directly in all but highly purified isolates, since cleavage at the sn.sup.2 position of phospholipids can also be the result from the combined sequential activities of PLA.sub.1 and lysophospholipase. As can be seen, however, many of these enzymes have been prepared from cells associated with inflammatory responses (i.e., macrophages, leukocytes, chondrocytes, synoviocytes, etc.) or inflammatory exudates. Nevertheless, the lack of cause/effect data has made it difficult to establish which, if any, of these enzymes are key in the inflammatory response.
The isolation of the PLA.sub.2 form responsible for rheumatoid arthritis in vivo would provide an important tool useful in the design of anti-inflammatory drugs. Based on the work with digestive and venom PLA.sub.2 inhibitors, it is believed that the form(s) of PLA.sub.2 responsible for inflammatory disease, while similar, are sufficiently different in structure such that inhibitors of digestive or venom PLA.sub.2 do not necessarily inhibit the latter form in vivo. Thus, to efficiently design specific inhibitors, it is necessary to isolate the specific PLA.sub.2 (s) that are involved in rheumatoid arthritis in sufficient quantity so that it can be structurally characterized. PLA.sub.2 is also generally useful in the food processing industry (Dutilh & Groger, 1981, J Sci Food Agric 32:451-458) and the preservation of fish. Mazeaud & Bilinski (1976) J Fish Res Board Can 33:1297-1302.